Advancing protein therapeutics through proximity-induced chemistry

Linqi Cheng Yixian Wang, Yiming Guo, Sophie S. Zhang Han Xiao
Cell Chemical Biology, 2024 Volume 31,  3, 428 - 445

https://doi.org/10.1016/j.chembiol.2023.09.004

Recent years have seen a remarkable growth in the field of protein-based medical treatments. Nevertheless, concerns have arisen regarding the cytotoxicity limitations, low affinity, potential immunogenicity, low stability, and challenges to modify these proteins. To overcome these obstacles, proximity-induced chemistry has emerged as a next-generation strategy for advancing protein therapeutics. This method allows site-specific modification of proteins with therapeutic agents, improving their effectiveness without extensive engineering. In addition, this innovative approach enables spatial control of the reaction based on proximity, facilitating the formation of irreversible covalent bonds between therapeutic proteins and their targets. This capability becomes particularly valuable in addressing challenges such as the low affinity frequently encountered between therapeutic proteins and their targets, as well as the limited availability of small molecules for specific protein targets. As a result, proximity-induced chemistry is reshaping the field of protein drug preparation and propelling the revolution in novel protein therapeutics.

Molecular Insights into the Impact of Mutations on the Binding Affinity of Targeted Covalent Inhibitors of BTK

 Ernest Awoonor-Williams and Abd Al-Aziz A. Abu-Saleh

The Journal of Physical Chemistry B 2024

DOI: 10.1021/acs.jpcb.4c00310

Targeted covalent inhibitors (TCIs) have witnessed a significant resurgence in recent years, particularly in the kinase drug discovery field for treating diverse clinical indications. The inhibition of Bruton’s tyrosine kinase (BTK) for treating B-cell cancers is a classic example where TCIs such as ibrutinib have had breakthroughs in targeted therapy. However, selectivity remains challenging, and the emergence of resistance mutations is a critical concern for clinical efficacy. Computational methods that can accurately predict the impact of mutations on inhibitor binding affinity could prove helpful in informing targeted approaches─providing insights into drug resistance mechanisms. In addition, such systems could help guide the systematic evaluation and impact of mutations in disease models for optimal experimental design. Here, we have employed in silico physics-based methods to understand the effects of mutations on the binding affinity and conformational dynamics of select TCIs of BTK. The TCIs studied include ibrutinib, acalabrutinib, and zanubrutinib─all of which are FDA-approved drugs for treating multiple forms of leukemia and lymphoma. Our results offer useful molecular insights into the structural determinants, thermodynamics, and conformational energies that impact ligand binding for this biological target of clinical relevance.

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1

Ryan D. Imhoff, Rishi Patel, Muhammad Hassan Safdar, Hannah B. L. Jones, Adan Pinto-Fernandez, Iolanda Vendrell, Hao Chen, Christine S. Muli, Aaron D. Krabill, Benedikt M. Kessler, Michael K. Wendt, Chittaranjan Das, and Daniel P. Flaherty

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01661

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations

Michael Westberg et al.

Sci. Transl. Med.16,eadi0979(2024).

DOI:10.1126/scitranslmed.adi0979

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D

Qinheng Zheng, Ziyang Zhang, Keelan Z. Guiley & Kevan M. Shokat

Nat Chem Biol 2024

 https://doi.org/10.1038/s41589-024-01565-w

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes

Daisuke Ogasawara, David Konrad, Zher Yin Tan, Kimberly Carey, Jessica Luo, Sang Joon Won, Haoxin Li, Trever Carter, Kristen DeMeester, Evert Njomen, Stuart Schreiber, Ramnik Xavier, Bruno Melillo, Benjamin Cravatt

Cell Chemical Biology, 2024

https://doi.org/10.1016/j.chembiol.2024.10.005

bioRxiv 2024.02.27.582206; 

doi: https://doi.org/10.1101/2024.02.27.582206

Chemical proteomics has emerged as a versatile approach to discover small-molecule ligands for proteins in native biological systems. Nonetheless, only a limited diversity of chemistry has so far been explored in chemical proteomic studies. In this study, we describe a set of DOS-inspired “photo-stereoprobes” that possess distinct physicochemical properties (e.g., greater three-dimensionality) compared to fragments. We identified >200 proteins showing stereoselective interactions with photo-stereoprobes in human cancer cells and confirmed many of these binding events with recombinant proteins. Interestingly, interactions that were not recapitulated in recombinant systems were enriched for proteins that are parts of large complexes, and additional stereoselective liganding events were identified exclusively in cells, but not cell lysates. These results highlight the value of chemical proteomic experiments that can map small-molecule interactions with endogenously expressed proteins in living systems. More broadly, the stereoprobe-liganded proteins were from diverse structural and functional classes, including adaptor/scaffolding and transcriptional regulatory proteins that have been historically challenging to target with small molecules. We further demonstrated that photo-stereoprobe-protein interactions can be converted into NanoBRET assays compatible with HTS to enable access to larger and more diverse small-molecule libraries. Finally, we demonstrated the utility of photo-stereoprobes in phenotypic screening by discovering compounds that stereoselectively modulate autophagy through engaging the mitochondrial protease CLPP. Taken together, our findings support the versatility of photo-stereoprobes as tools to expand the ligandability of the human proteome.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism

VANHA N. PHAM, KEVIN J. BRUEMMER, JOEL D. W. TOH, EVA J. GE, LOGAN TENNEY, CARL C. WARD, FELIX A. DINGLER, CHRISTOPHER L. MILLINGTON, CARLOS A. GARCIA-PRIETO  MIA C. PULOS-HOLMES  NICHOLAS T. INGOLIA LUCAS B. PONTEL MANEL ESTELLER  KETAN J. PATEL  DANIEL K. NOMURA AND CHRISTOPHER J. CHANG 

Science 2023 382, 6670

DOI: 10.1126/science.abp9201

INTRODUCTION

One-carbon metabolism manages cellular carbon pools by detoxifying highly reactive carbon species, such as aldehydes, and diverting their carbon toward the biosynthesis of useful products, including amino acids and nucleotides. Formaldehyde (FA) is a major one-carbon unit derived from exogenous environmental exposure and endogenous sources and is quickly scavenged in the cell through enzymatic oxidation to formate and carbon dioxide and/or metabolized through the folate cycle. S-adenosylmethionine (SAM) serves as the primary cellular methyl donor and harbors one-carbon units in a stable and accessible form. The ability to decipher the biochemical interplay between toxic reactive carbon species and stable physiological carbon units is essential for understanding fundamentals of one-carbon metabolism across all kingdoms of life. Especially important is understanding how aberrant carbon imbalances are connected to human diseases such as cancer, liver diseases, and asthma. Although the chronic exposure of toxic aldehydes is correlated to disease states, biological mechanisms of aldehyde signaling and their relation to carbon metabolism remain underexplored.

RATIONALE

Owing to its highly electrophilic nature, we hypothesized that FA could act as a one-carbon signal sensed by privileged cysteine sites across the proteome. FA reacts with cysteines on synthetic peptides, and we designed an unbiased, proteome-wide profiling study to systematically identify FA-sensitive cysteine residues. This work builds a biochemical framework for understanding global FA reactivity as a selective posttranslational modification of target proteins and downstream regulatory effects of such modifications.

RESULTS

Activity-based protein profiling identified FA modification of privileged cysteine sites across the proteome, including several enzymes responsible for FA metabolism, one-carbon metabolism, and amino acid biosynthesis. We focused on biochemical characterization of a key Cys120 residue on the SAM-generating enzyme S-adenosylmethionine synthase isoform type-1 (MAT1A) that is proximal to the MAT1A active site. FA exposure resulted in inhibition of MAT1A activity in an isoform-specific manner, which led to decreased SAM production. Cellular models containing only the MAT1A isoform displayed a reciprocal decrease in SAM levels with increasing doses of FA exposure. Moreover, an Adh5–/– mouse model of chronic FA elevation also showed SAM deficiency accompanied by lower levels of methylation on select histone methyl sinks. The chronic FA model also resulted in a decrease in methylation of the Mat1a promoter region, resulting in increased MAT1A expression as a compensatory mechanism to maintain available carbon units. We deciphered a compensatory biochemical feedback cycle where FA-dependent SAM deficiency led to an increase in MAT1A expression through genetic and epigenetic mechanisms regulated by FA-dependent transcription factors and DNA promoter hypomethylation, respectively.

CONCLUSION

In contrast to the traditional view of FA as an indiscriminate electrophile and toxic metabolite, we show that FA is sensed by specific cysteine sites in the proteome to regulate one-carbon metabolism feedback cycles through SAM biosynthesis. FA reacts with a key cysteine residue on MAT1A to inhibit its activity, resulting in SAM depletion and downstream changes in histone and DNA methylation. Under normal homeostatic conditions, FA is quickly sequestered into the folate cycle for conservation of one-carbon units to maintain balanced SAM biosynthesis. In response to FA overload, reciprocal SAM depletion through isoform-specific MAT1A inhibition results in changes to cellular methylation potential, epigenetic dysregulation, and perturbations in one-carbon metabolism, which in turns leads to compensatory up-regulation of MAT1A expression. This work provides a starting point for further exploration of aldehydes as signaling agents and the nexus between one-carbon metabolism and one-carbon signaling.