Drugging the undruggable: Advances in targeting KRAS signaling in solid tumors.

Cancer remains the leading cause of global mortality, prompting a paradigm shift in its treatment and outcomes with the advent of targeted therapies. Among the most prevalent mutations in RAS-driven cancers, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for approximately 86% of cases worldwide, particularly in lung, pancreatic, and colon cancers, contributing to poor prognosis and reduced overall survival. Despite numerous efforts to understand the biology of KRAS mutants and their pivotal role in cancer development, the lack of well-defined drug-binding pockets has deemed KRAS an "undruggable" therapeutic target, presenting significant challenges for researchers and clinicians alike. Through significant biochemical and technological advances, the last decade has witnessed promising breakthroughs in targeted therapies for KRAS-mutated lung, colon, and pancreatic cancers, marking a critical turning point in the field. In this chapter, we provide an overview of the characteristics of KRAS mutations across various solid tumors, highlighting ongoing cutting-edge research on the immune microenvironment, the development of KRAS-driven mice models, and the recent progress in the exploration of specific KRAS mutant-targeted therapeutic approaches. By comprehensive understanding of the intricacies of KRAS signaling in solid tumors and the latest therapeutic developments, this chapter will shed light on the potential for novel therapeutic strategies to combat KRAS-driven tumors and improve patient outcomes.

Role of a substrate binding pocket in the amino terminal domain of Mycobacterium tuberculosis caseinolytic protease B (ClpB) in its function.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis when infects the host encounters several stresses within the host, resulting in aggregation of its proteins. To resolve this problem Mtb uses chaperones to either repair the damage or degrade the aggregated proteins. Mtb caseinolytic protein B (ClpB) helps in the prevention of aggregation and also resolubilization of aggregated proteins in bacteria, which is important for the survival of Mtb in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of Mtb ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of Mtb ClpB in silico. A substrate binding pocket, within the NTD of ClpB comprising of residues L136, R137, E138, K142, R144, R148, V149, Y158, and Y162 forming an ɑ-helix was thus identified. The residues L136 and R137 of the ɑ-helix were found to be important for the interaction of DnaK to ClpB. Further, nine single alanine recombinant variants of the identified residues were generated. As compared to the wild-type Mtb ClpB all the Mtb ClpB variants generated in this study were found to have reduced ATPase and protein refolding activity indicating the importance of the substrate binding pocket in ClpB function. The study demonstrates that the NTD of Mtb ClpB is important for its substrate interaction activity, and the substrate binding pocket identified in this study plays a crucial role in this interaction.Communicated by Ramaswamy H. Sarma.

ClpB is an essential stress regulator of Mycobacterium tuberculosis and endows survival advantage to dormant bacilli.

The ability to tolerate multiple host derived stresses, resist eradication and persist within the infected individuals is central to the pathogenicity of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Mycobacterial survival is contingent upon sensing environmental perturbations and initiating a fitting response to counter them. Therefore, understanding of molecular mechanisms underlying stress tolerance and sensing in Mtb is critical for devising strategies for TB control. Our study aims to delineate the role of ClpB, a heat shock protein of Hsp100 family, in the general stress response and persistence mechanisms of Mtb. We demonstrate that Mtb requires ClpB to survive under stressful conditions. Additionally, we show that ClpB is necessary for the bacteria to persist in latency-like conditions such as prolonged hypoxia and nutrient-starvation. The disruption of ClpB results in aberrant cellular morphology, impaired biofilm formation and reduced infectivity of Mtb ex vivo. Our study also reports an alternative role of ClpB as a chaperokine which elicits inflammatory response in host. We conclude that ClpB is essential for Mtb to survive within macrophages, and plays a crucial part in the maintenance of dormant Mtb bacilli in latent state. The absence of ClpB in human genome makes it an attractive choice as drug target for TB.

Mechanism of HrcA function in heat shock regulation in Mycobacterium tuberculosis.

Molecular chaperones are a conserved family of proteins that are over-expressed in response to heat and other stresses. The regulation of expression of chaperone proteins plays a vital role in pathogenesis of various bacterial pathogens. In M. tuberculosis, HrcA and HspR negatively regulate heat shock protein operons by binding to their cognate DNA elements, CIRCE and HAIR respectively. In this study, we show that M. tuberculosis HrcA is able to bind to its cognate CIRCE DNA element present in the upstream regions of groES and groEL2 operons only with the help of other protein(s). It is also demonstrated that M. tuberculosis HrcA binds to a CIRCE like DNA element present in the upstream region of hrcA gene suggesting its auto-regulatory nature. In addition, we report the presence of a putative HAIR element in the upstream region of groES operon and demonstrate the binding of HspR to it. In vitro, HrcA inhibited the DNA binding activity of HspR in a dose-dependent manner. The current study demonstrates that M. tuberculosis HrcA requires other protein(s) to function, and the heat shock protein expression in M. tuberculosis is negatively regulated jointly by HrcA and HspR.

Dicarboxylate Transporters of Azospirillum brasilense Sp7 Play an Important Role in the Colonization of Finger Millet (Eleusine coracana) Roots.

Azospirillum brasilense is a plant growth-promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

The amino-terminal domain of Mycobacterium tuberculosis ClpB protein plays a crucial role in its substrate disaggregation activity.

Mycobacterium tuberculosis (Mtb) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved molecular chaperone machinery. ClpB, a unique member of the AAA+ family of chaperones, is responsible for resolving aggregates in Mtb and many other bacterial pathogens. Mtb produces two isoforms of ClpB, a full length and an N-terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N-terminal domain (NTD) of Mtb ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of Mtb ClpB. We found the NTD to be dispensable for oligomerization, ATPase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the NTD of Mtb ClpB has a hydrophobic groove that contains four hydrophobic residues: L97, L101, F140 and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.

A ribonuclease inhibitor resistant dimer of human pancreatic ribonuclease displays specific antitumor activity.

Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.